Human immunodeficiency virus type 1 infection requires reverse transcription of nascent viral RNA

We have previously shown that during human immunodeficiency virus type 1 (HIV-1) infection in vitro continued reverse transcription is required for stable HIV-1 production, but entry by progeny virus is not. To determine the source of the viral RNA reverse-transcribed late in infection, we employed inhibitors of HIV-1 transmission, reverse transcription, and proteolysis of the Gag-Pol polyprotein to interrupt HIV-1 infection in vitro. The kinetics of synthesis of viral DNA, RNA, and proteins was examined. During single-cycle infection, inhibition of reverse transcription 24-72 hr after infection delayed production of viral RNA and protein 10 days. Although viral DNA was detected in Southern blots, inhibition of Gag-Pol processing or transient inhibition of reverse transcription blocked its expression. We propose that after initial reverse transcription of input virion RNA is complete, newly synthesized HIV-1 RNA is reverse-transcribed before its export in virions to yield the viral DNA required for stable HIV-1 production.